How to Tri-Culture Human Pluripotent Stem Cell (hPSC)-Derived Forebrain Neurons, Astrocytes, and Microglia

Brain tissue is comprised of neuronal and glial cell types that are in continuous communication to maintain neural circuitry and tissue homeostasis. In vitro, human pluripotent stem cell (hPSC)-derived models provide an opportunity to generate pure populations of these various cell types from the same genetic background and recombine them more precisely than the mixed populations that could be obtained from primary tissues. Such controlled co-cultures can be used to model neuronal-glial and neuroimmune interactions. This protocol describes a method to tri-culture hPSC-derived microglia, astrocytes, and forebrain neurons by generating each cell type separately and then combining them under optimized culture conditions.

Important Notes:

  • This protocol involves differentiation of hPSCs to microglia using STEMdiff™ Hematopoietic Kit followed by STEMdiff™ Microglia Differentiation Kit. In parallel, hPSCs are also differentiated to astrocytes using STEMdiff™ Astrocyte Differentiation Kit and matured for at least 3 weeks with STEMdiff™ Astrocyte Maturation Kit. Alongside the first two cell types, hPSCs are differentiated to forebrain neurons using STEMdiff™ Forebrain Neuron Differentiation Kit and matured for at least 7 days with STEMdiff™ Forebrain Neuron Maturation Kit. The matured forebrain neurons are first combined with astrocytes in co-culture. Microglia are subsequently added to the co-culture to generate a tri-culture consisting of all three cell types.
  • There are multiple workflow options to generate the three hPSC-derived cell types in parallel prior to combining them in co-culture. For details, please refer to the relevant Product Information Sheets (PIS’s).
  • If desired, forebrain neuron or astrocyte maturation may be extended for longer than 7 days or 3 weeks, respectively. Adjust Part I and Part III of this protocol to begin earlier as needed.
  • Cells may be cryopreserved at various points for increased flexibility in co-culture planning (please contact techsupport@stemcell.com for instructions).
  • The recommended tri-culture period is 3 - 10 days. To ensure optimal microglia survival, do not exceed 10 days of culture following the 24-day microglia differentiation (i.e. steps 1 - 8 in section A under “Directions for Use” in the PIS for STEMdiff™ Microglia Differentiation Kit).
  • To co-culture forebrain neurons and astrocytes without microglia, refer to this optimized protocol. To co-culture forebrain neurons and microglia without astrocytes, refer to this optimized protocol.

Materials

*Required for microglia differentiation as outlined in the PISs for STEMdiff™ Hematopoietic Kit and for STEMdiff™ Microglia kits.
✝Required for forebrain neuron differentiation as outlined in the PIS for STEMdiff™ Forebrain Neuron Kits.
^Required for astrocyte differentiation as outlined in the PIS for STEMdiff™ Astrocyte Kits.

Part I: Differentiate hPSCs to Astrocytes

  1. Follow either the embryoid body (EB) or monolayer protocol outlined in the STEMdiff™ Astrocyte Differentiation and Maturation Kit PIS (Document #10000006879).
  2. Continue the astrocyte maturation phase until astrocytes have been cultured in STEMdiff™ Astrocyte Maturation Medium for at least 3 weeks.
  3. Verify successful astrocyte differentiation by performing immunocytochemistry on a subset of cultured cells. With conventional use of the STEMdiff™ Astrocyte System, the cell population should be > 70% S100ꞵ+, > 60% GFAP+, and < 15% positive for ꞵIII-tubulin or doublecortin (DCX).

Part II: Differentiate hPSCs to Microglia

  1. Generate hematopoietic progenitor cells (HPCs) by following the entire protocol outlined in the STEMdiff™ Hematopoietic Kit PIS (Document #10000003456).
  2. Evaluate the efficiency of HPC differentiation by flow cytometry. The resulting cell population should be > 90% CD43+ and > 20% positive for coexpression of CD34/CD45.
  3. Follow steps 1 - 8 outlined in Section A (Microglia Differentiation) under “Directions for Use” in the STEMdiff™ Microglia Differentiation & Maturation Kit PIS (Document #10000006003).
  4. Evaluate the efficiency of microglia differentiation by flow cytometry. The resulting cell population should be > 80% positive for coexpression of CD45 and CD11b.
  5. Optional: Microglia may be further cultured using STEMdiff™ Microglia Maturation Kit (Catalog #100-0020) prior to tri-culture setup in Part V. However, do not exceed 10 days in total of additional culture time after microglia differentiation (total of maturation and co-culture period combined).

Part III: Differentiate hPSCs to Forebrain Neurons

  1. Follow either the embryoid body (EB) or monolayer protocol outlined in the STEMdiff™ Forebrain Neuron Differentiation and Maturation Kit PIS (Document #10000005464).
    Note: When seeding neuronal precursors into STEMdiff™ Forebrain Neuron Maturation Medium (section C, step 1 under “Directions for Use” in the PIS), the suggested density for tri-culture with astrocytes and microglia ranges from 1.5 x 104 - 4 x 104 cells/cm2. The optimal density should be determined by the user.
  2. Continue the forebrain neuron maturation phase until neurons have been cultured in STEMdiff™ Forebrain Neuron Maturation Medium for at least 7 days.
  3. Verify successful forebrain neuron differentiation by performing immunocytochemistry on a subset of cultured cells. The resulting cell population should be > 90% positive for ꞵIII-tubulin and FOXG1, and < 10% positive for the astrocyte marker GFAP.

Part IV: Set Up Forebrain Neuron-Astrocyte Co-Culture

  1. Dissociate mature astrocytes from Part I according to steps 1 - 5 of section C (Astrocyte Maturation) of the STEMdiff™ Astrocyte PIS (Document #10000006879).
    Note: Resuspend the cells in 1 mL of complete STEMdiff™ Astrocyte Maturation Medium and perform a cell count using Trypan Blue and a hemocytometer.
  2. Dilute the cell suspension in additional complete STEMdiff™ Astrocyte Maturation Medium to obtain the required cell concentration and volume.
    Note: The concentration and volume of the astrocyte suspension should be optimized by the user and will depend on the desired astrocyte-to-neuron cell-type ratio, the number of culture wells used, and the initial cell density of forebrain neuronal precursors plated in Part III, step 1. The recommended astrocyte-to-forebrain neuron cell-type ratio for tri-culture is 1:1.
  3. Remove and discard the culture medium from the forebrain neurons generated in Part III.
  4. Seed the resuspended astrocytes prepared in step 2 onto the forebrain neurons and maintain overnight in STEMdiff™ Astrocyte Maturation Medium. Proceed to Part V.

Part V: Prepare Optimized Microglia-Forebrain Neuron-Astrocyte Tri-Culture Medium and Set Up Tri-Culture

  1. In the PIS for BrainPhys™ (Document #10000000225), follow the instructions under “Preparation of Complete Differentiation Medium” (section B) to prepare the required volume of BrainPhys™ Neuronal Medium + supplements.
    Note: The required volume of medium will depend on the culture plate format and desired length of the tri-culture period.
  2. Thaw STEMdiff™ Microglia Supplement 2 (Component #100-0023 of STEMdiff™ Microglia Differentiation Kit) at room temperature (15 - 25°C) until just thawed, or alternatively at 2 - 8°C overnight. Mix thoroughly.
    Note: If not used immediately, aliquot supplement and store at -20°C. Do not exceed the expiry date (EXP) as indicated on the label.
    Note: If you do not have enough leftover STEMdiff™ Microglia Supplement 2 to prepare the optimized tri-culture medium, please contact techsupport@stemcell.com to purchase this component individually.
  3. Prepare optimized Tri-Culture Medium as follows:
    a. Add STEMdiff™ Microglia Supplement 2 to BrainPhys™ Neuronal Medium + supplements to a final concentration of 1X (e.g. 40 µL of supplement per 10 mL of medium).
    b. Mix thoroughly.
    Note: If not used immediately, store Tri-Culture Medium at 2 - 8°C for up to 2 weeks.
  4. Collect microglia from Part II:
    a. Transfer the entire cell suspension to a 15 mL conical tube. To ensure all cells are collected, additional washes may be performed using warm DMEM/F-12 with 15 mM HEPES.
    b. Centrifuge at 300 x g for 5 minutes.
    c. Remove supernatant and resuspend the pellet in an appropriate volume of Tri-Culture Medium.
    d. Count cells using Trypan Blue and a hemocytometer.
  5. Dilute the microglia cell suspension in additional Tri-Culture Medium to obtain the required final cell concentration and volume.
  6. Microglia Seeding Density Considerations:
  7. Remove and discard the culture medium from the forebrain neuron-astrocyte co-cultures generated in Part IV.
  8. Seed the resuspended microglia onto the co-cultured forebrain neurons and astrocytes.
  9. Place the plate in a 37°C, 5% CO2 incubator. Distribute cells evenly by moving the plate in several quick, short, back-and-forth and side-to-side motions.
  10. Perform a half-medium change every 2 - 3 days with Tri-Culture Medium.
    Note: Microglia are semi-adherent and may unintentionally be removed during medium changes. Prior to performing half medium changes, centrifuge the plate in a swinging-bucket centrifuge with plate adaptors at 100 x g for 2 minutes to force the cells to settle.

Characterize by Immunocytochemistry

  • Immunocytochemistry may be performed after 3 - 10 days of tri-culture, using the following primary antibodies:
    • Neuronal marker: Anti-Beta-Tubulin III Antibody, Clone TUJ1 (Catalog #60052)
    • Microglia marker: Rabbit polyclonal anti-IBA1 (Synaptic Systems Catalog #234 003)
    • Astrocyte marker: Chicken polyclonal anti-GFAP (Aves Labs Catalog #GFAP)
  • Total cell density may be assessed by DAPI staining.
  • Expected observations: Cultures should consist of healthy microglia integrated among forebrain neurons and astrocytes. Microglia should display an unactivated, ramified morphology with extended processes.
  • Document #PR00065
  • Version 1.0.0
  • August 2022


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